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Synthesis of Lactam-Bridged and Lipidated Cyclo-Peptides As Promising Anti-Phytopathogenic Agents

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Synthesis of Lactam-Bridged and Lipidated Cyclo-Peptides As Promising Anti-Phytopathogenic Agents molecules Article Synthesis of Lactam-Bridged and Lipidated Cyclo-Peptides as Promising Anti-Phytopathogenic Agents Aldrin V. Vasco 1 , Martina Brode 1, Yanira Méndez 1,2, Oscar Valdés 3 , Daniel G. Rivera 1,2,* and Ludger A. Wessjohann 1,* 1 Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany; [email protected] (A.V.V.); [email protected] (M.B.); [email protected] (Y.M.) 2 Center for Natural Products Research, Faculty of Chemistry, University of Havana, Zapata y G, Havana 10400, Cuba 3 Vicerrectoría de Investigación y Postgrado, Universidad Católica del Maule, Talca 3460000, Chile; [email protected] * Correspondence: [email protected] (D.G.R.); [email protected] (L.A.W.); Tel.: +53-7879-2331 (D.G.R.); +49-345-5582-1301 (L.A.W.) Academic Editors: Alexander Dömling and Shabnam Shaabani Received: 11 November 2019; Accepted: 7 February 2020; Published: 13 February 2020 Abstract: Antimicrobial resistance to conventional antibiotics and the limited alternatives to combat plant-threatening pathogens are worldwide problems. Antibiotic lipopeptides exert remarkable membrane activity, which usually is not prone to fast resistance formation, and often show organism-type selectivity. Additional modes of action commonly complement the bioactivity profiles of such compounds. The present work describes a multicomponent-based methodology for the synthesis of cyclic polycationic lipopeptides with stabilized helical structures. The protocol comprises an on solid support Ugi-4-component macrocyclization in the presence of a lipidic isocyanide. Circular dichroism was employed to study the influence of both macrocyclization and lipidation on the amphiphilic helical structure in water and micellar media. First bioactivity studies against model phytopathogens demonstrated a positive effect of the lipidation on the antimicrobial activity. Keywords: peptide cyclization; antimicrobial peptides (AMPs); multicomponent reactions (MCRs); lipopeptides; fungicides; antimycotics; plant pathogens 1. Introduction The continuous incidence of bacterial and fungal resistance has aroused great attention and re-sparked the interest in discovering or designing alternative antimicrobial substances that can be used for various applications including clinical uses as well as the preservation of food and dairy products [1–3]. Phytopathogenic microorganisms affect not only the yield of crop plants or stored foods (e.g., fruits) but also produce toxins, which have been described to be potentially harmful to the health of consumers and animals, including the induction of cancer, immunosuppression, and growth disorders [4–6]. Furthermore, the development of environmentally friendly and resistance-withstanding alternatives to the currently used pesticides remains one of the biggest challenges for plant scientists [7]. Polycationic antimicrobial peptides (AMPs) are essential components in the innate immune system of all multicellular organisms [8–10]. Even when the mechanisms of action of AMPs are not fully understood, in most cases their biological effects are believed to involve membrane disruption of the target cells by either acting through a detergent-like disruption or by the formation of transient transmembrane pores [11–13]. Molecules 2020, 25, 811; doi:10.3390/molecules25040811 www.mdpi.com/journal/molecules Molecules 2020, 25, 811 2 of 14 An increasing amount of articles describing the use of AMPs as antimycotic agents—including phytopathogenic fungi—has been published in the last few years [3,7,14–18]. Due to the eukaryotic nature of fungal cells, it is not an easy task to develop antifungal (antimycotic) drugs which do not exhibit lytic activity against plant and human cells [19]. Antimicrobial peptides, including antimicrobial biosurfactants, have been reported to act as both narrow and broad-spectrum agents against Gram-positive and Gram-negative bacteria, or fungi [20]. Ligation of AMPs to lipidic chains has proved effective for the design of broad-spectrum antifungal peptides with a low hemolytic activity, which can be designed to exhibit their bioactivity in a pH-dependent manner [8]. Lipidation of the peptide chain is broadly present in nature and it is a structural feature of several biosurfactants such as the Surfactin [21], Iturin [22], and Fengycin [23] families. These families of Bacillus lipopeptides have shown antagonistic activities towards a wide range of phytopathogens including fungi, bacteria and oomycetes [15]. Lipopeptides are produced non-ribosomally in bacteria, yeasts and fungi, and can act either over specific targets or by disrupting the cellular membrane. Subsequently, this type of antimicrobials has the advantage that it takes several hundred generations at low concentrations to develop bacterial resistance, a reason why they are widely considered as potential alternatives to the growing problem of resistance to conventional (protein targeting) antibiotics [3,15]. Our group took advantage of the high efficiency and atom economy of multicomponent reactions (MCRs) for macrocyclization and simultaneous peptide ligation and modification [24,25], e.g., for lipidation [26–28], glycosylation [29], pegylation and labeling [30]. We have shown how MCRs can be useful for the design of cyclic lipopeptide analogs of naturally-occurring biosurfactants and cytotoxins [31]. In a recent report, we developed a solid-supported Ugi-based stapling protocol for the convenient functionalization of short helical peptides [30]. Contemporary stapling protocols—mostly based on alkene metathesis [32,33], lactam bridge formation [34–36], CuI-catalyzed alkyne-azide cycloaddition (click) [37], Lys-N"- and Cys-S-arylations [38–40], and Cys-alkylation [41,42]—comprise the common stabilization methods for helical secondary structures by (side) chain-to-side chain tethering of two amino acids on the same face of an ideal helix, i.e., between amino acids at positions i i + 4, i i + 7, or i i + 11 within the peptidic sequence. Likewise, the Ugi-based approach allows the ! ! ! helical stabilization by reacting side chains from Lys and Glu/Asp amino acids at i i + 4 positions while ! incorporating the moiety arising from the isocyanide component as an additional functionalization at the resulting tertiary lactam bridge [30]. Herein we present the synthesis of two series of helical polycationic cyclic lipopeptides designed to possess a facial amphiphilic character. We envisioned that the introduction of a lipid moiety at the closing lactam bridge opposite to the polycationic face of the peptide—achievable through a Ugi-based multicomponent stapling approach—should stabilize the helical structure of these peptides and simultaneously enhance their amphiphilic character. To assess how these structural modifications can influence the bioactivity profile of polycationic lipopeptides, we sought to evaluate the activity of the compounds against three crop-affecting phytopathogens, which are also standard pathogens in industrial plant protection screenings. 2. Results and Discussion To evaluate the suitability of the simultaneous macrocyclization and N-lipidation achievable by the Ugi-stapling methodology for the design of lactam-bridged N-lipidated peptides with antifungal activity, two sets of helical amphiphilic peptides were prepared. We aimed at evaluating how the lipidation at the lactam bridge affects the bioactivity of polycationic helical peptides as compared with their non-lipidated lactam-bridged analogs. 2.1. Synthesis of Peptides Based on the Ac-LAKLLKAKAKAD-NH2 Sequence As a starting point for our study, we decided to design a peptide with 12 amino acids in its sequence—which is equivalent to three turns of an ideal α-helix—the facial amphiphilicity of which could arise from the distribution of cationic and lipophilic side chains at one vs. the other face of Molecules 2020, 25, 811 3 of 14 the helix, respectively. This design has previously proven effective to stabilize helical structures when Ugi-stapling between residues 5 and 9 is conducted [30]. Herein we intended to perform the cyclization at the N-terminus, envisioning that in this way the interaction of a lipid functionalization with the hydrophobic face of the helix would be higher and sterically less disturbing than when the stapling is situated in the middle of the sequence, resulting in a peptide with better hydrophobicity / amphiphilicity, which is important for membrane activity. Resin-bound, fully-protected peptide 1 was prepared on solid support using a classical Fmoc/tBu strategy (Scheme1A), with the sequential incorporation of the Fmoc-protected amino acids and final N-terminal acetylation. To achieve the cyclic peptides 2, 3 and 4, a third dimension of orthogonality was inserted by Alloc and Allyl ester-protected Lys and Asp at positions 8 and 12, respectively. This enables the selective deprotection of these side chains by treatment with tetrakis(triphenylphosphine)palladium(0) and phenylsilane under a stream of nitrogen, thus leading to the resin-bound peptide 1. The unprotected side chains can then be employed in the desired on-resin macrocyclization protocol. In this way, cyclic peptide 4 was prepared from resin-bound peptide 1 by lactam-bridge formation in the presence of PyAOP as an activating agent. Even when a resin with relatively low loading was employed, 20% of dimerization product was observed in the crude peptide by UHPLC-MS analysis. The simultaneous cleavage from the resin and Boc/tBu side chain
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